The Construction Of Mycobacterial Membrane-anchored Expression Vector And Analysis Of Sub-cellualr Localization Of Target Protein

TB (Tuberculosis) caused by Mycobacterium tuberculosis (M. tb) is a contagious disease, and BCG (bacillus Calmette-Guérin) is recognized worldwide as the only effective TB vaccine. One of the important ways to improve the effectiveness of TB vaccines is to develop the recombinant BCG. Under normal circumstances, constructed rBCG secreting antigens or anchored to the extracellular surface of the cell membrane, can be more effective in presenting and immune response. Objective To construct Mycobacterial membrane-anchored expression vector and to analyze expression level and sub-cellualr localization of exogenous target protein. Methods The mutants of the mutated Mycobacterium tuberculosis (M. tb) furA gene promoter (pfurAma) were obtained by direct mutagenesis of the pfurAma sequence, and these resulting mutated and the prototype pfurAma were then cloned into the probe vector pMC210 respectively, and gene-fused to the immediately upstream of the lacZ reporter to analyze their transcrioption activities in M. smegmatis. The optimal promoter (pfurAma-N) was screened by theβ-galactosidase assay, and then was used to develop a Mycobacterial intracellular expression vector pMFA42 by changing the phsp60 of the E. coli-mycobacteiral shuttle plasmid pMV261. Based on the vector pMFA42 which contained the strongest promoter of pfurAma-N, the signal sequence of M. tb 19 kDa lipoprotein (19ss) was synthesized and was then cloned into the downstream of pfurAma-N to generate the Mycobacterial membrane-anchored expression vector pMFA42M. The coding gene of enhanced green fluorescent protein (EGFP) was amplified by PCR, and then sub-cloned into these two vectors described above to construct recombinant EGFP fused and membrane-anchored strains, respectively. The coding genes of M. tb immuno-dominant antigens Ag85A and its chimera Ag856A2 were then sub-cloned intothe membrane-anchored construct pMFA42M to produce recombinant M. tb antigen-EGFP fused-expression strains. After that, expression levels and sub-cellualr localization of exogenous target protein were further analyzed by Western-blot and flow cytometry sorting (FCS), and the fluorescence intensities of recombinant EGFP-expressed strains were observed in vitro directly and after transfection of murine macrophage cell line RAW264.7. Results The pfurAma-N showed the most competitive activity about two times than phsp60, yet pfurAma-N3 declined half of the phsp60 activity result from the three bases longer distance from the initiator codon to its RBS. The novel Mycobacterial membrane-anchored expression vector was constructed successfully by introduction of signal sequence of M. tb 19 kDa lipoprotein. Using of EGFP as model antigen, exogenous target protein was demonstrated to express with high level and could be anchored into cell membrane of recombinant mycobaterial strains. Conclusion A novel Mycobacterial membrane-anchored expression vector was constructed successfully to research recombinant BCG and functions of Mycobacterial membrane proteins, and the constructed EGFP-expressed recombinant strains could be used to research cytophagy in cell model and Mycobacterial colony and translocation in animal immunization as a model indicator bacteria.